Molecular imaging has evolved into a discipline in its own right with applications to numerous areas of cancer biology and clinical medicine. Protein-protein interactions are at the heart of many biological processes including normal and diseased tissues. The long-term goal of this competitive renewal application is to discover and validate novel cancer drugs that target specific protein-protein interactions in living subjects, using optical imaging in conjunction with positron emission tomography (PET), computed tomography (CT), ultrasound (US) and other emerging clinical modalities. Addition goals also include that reporter gene technologies can be optimized for studying fundamental molecular/cellular events, non-invasively and repetitively in living subjects. Excellent progress has been made in this regard over the last-5 year funding period. We have developed and continued to optimize bioluminescence resonance energy transfer (BRET) strategies for imaging fewer number of cells in deeper tissues in small living subjects. We have also helped advance molecular imaging for drug discovery by development and validation of split reporter gene technologies for small animal imaging, including studying of heat shock protein 90 (Hsp90)/co-chaperone p23 interactions in cancer. The specific aims of the current renewal are to fully utilize the developed approaches as tool boxes for systems imaging of intracellular networks in small living subjects in response to drug treatment. In Aim 1 we will optimize and compare the sensitivity, specificity, dynamic range of BRET with split luciferase protein-assisted-complementation (PAC)-based strategies for imaging drug-modulated Hsp90/p23 interactions in small living subjects. In Aim 2 we will utilize the optimized assay from Aim 1 to screen large chemical compound libraries to identify new drugs that can disrupt Hsp90/p23 interactions by optical imaging in cell culture and tumor models using the same unified system, followed by mechanistic validation with standard biochemical studies. The downstream effects on glucose metabolism/DNA synthesis and blood-flow in tumor models will be monitored by PET/CT and US imaging, respectively. In Aim 3, we will validate the mechanisms and efficacies of novel histone deacetylase (HDAC) inhibitors in both cell culture and tumor models using the optimized system from Aim 1. We will also determine and validate the synergistic/additive effects of novel HDAC inhibitors in combination with the novel Hsp90 inhibitors (Aim 2) on disruption of Hsp90/p23 interactions, followed by monitoring the downstream effects in living subjects, using the same imaging approaches and biochemical studies as in Aim 2. The significance of the proposed work includes the establishment of a new paradigm for imaging intracellular communication networks pertaining to drug development. This will lead to many applications, especially strategies to image the interaction of drugs designed to inhibit disease-specific protein-protein interactions in living subjects. This will facilitate drug discovery and accelerate the transition of pre-clinical results to human clinical applications.